Prostate cancer incidence and mortality continue to be significant clinical problems, and the development of new treatment strategies depends on improving our basic understanding of prostate cancer biology. Such basic investigations in turn are dependent on accurate and relevant experimental models, both in vitro and in vivo. However, basic prostate cancer research has been challenged by difficulties in establishing patient- derived cell lines and the unavailability of adequately tailored 3D cell culture matrices. Currently, the majority of human prostate cancer cell lines are derived from castration-resistant metastases, and in contrast to other cancers such as pancreatic cancer, high-grade prostate tumors derived from clinical specimens do not efficiently establish in animal models as xenografts. Current 3D cell culture matrices such as Matrigel have enabled the field to establish some prostate cancer organoid cultures, but Matrigel?s usefulness is limited by its poor batch-to-batch consistency and difficulties in adjusting its composition for specific cell types and specific contexts. Thus, the development of more defined and customizable matrices would overcome a major technical hurdle in establishing and maintaining prostate cancer cell organoids for basic study. The Collier lab has recently developed a strategy for creating gel materials from defined sets of expressed proteins, using molecular self-assembly. In this approach, proteins are engineered to display a novel tag that allows bacteriologic expression in a monomeric state, but subsequent self-assembly into defined 3D culture matrices upon mixing with short synthetic peptides. These materials can be formulated into compositionally defined cell culture materials, allowing us in the proposed work to engineer improved 3D culture media that support prostate cancer organoid growth. The work will be conducted through two aims, as follows. Aim 1: Develop a tailored cell culture matrix for prostate cancer cell growth in vitro and tumor initiation in vivo, using novel self-assembling proteins (beta-Tails) and self-assembling peptides. Aim 2: Develop prostate-specific antigen (PSA)-responsive 3D culture media and engineer them to accelerate primary human prostate cell growth by releasing FGF10 as the cells secrete PSA. We expect these customizable cell culture matrices to be useful not only for culturing patient-derived prostate cancer cells, but also for a wide variety of additional cell types. This project will be a critical proof-of-concept that will establish the foundation of these materials for future development in a range of different culture contexts. The two PIs, Drs. Collier and Vander Griend, are appointed in the same department (Surgery), facilitating this collaboration aimed at solving the longstanding problem of unsuitable matrices for prostate organoid culture.